Soybean variety 93B47

ABSTRACT

A soybean variety designated 93B47, the plants and seeds of soybean variety 93B47, methods for producing a soybean plant produced by crossing the variety 93B47 with itself or with another soybean plant, and hybrid soybean seeds and plants produced by crossing the variety 93B47 with another soybean line or plant, and the creation of variants by mutagenesis or transformation of variety 93B47. This invention also relates to methods for producing other soybean varieties or breeding lines derived from soybean variety 93B47 and to soybean varieties or breeding lines produced by those methods.

FIELD OF THE INVENTION

This invention is in the field of soybean breeding, specificallyrelating to a soybean variety designated 93B47.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive soybean variety,designated 93B47 which has been the result of years of careful breedingand selection as part of a soybean breeding program. There are numeroussteps in the development of any novel, desirable plant germplasm. Plantbreeding begins with the analysis and definition of problems andweaknesses of the current germplasm, the establishment of program goals,and the definition of specific breeding objectives. The next step isselection of germplasm that possess the traits to meet the programgoals. The goal is to combine in a single variety an improvedcombination of desirable traits from the parental germplasm. Theseimportant traits may include higher seed yield, resistance to diseasesand insects, tolerance to drought and heat, and better agronomicqualities.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant. Soybean plants (Glucine max), are recognized to benaturally self-pollinated plants which, while capable of undergoingcross-pollination, rarely do so in nature. Insects are reported by someresearchers to carry pollen from one soybean plant to another and itgenerally is estimated that less than one percent of soybean seed formedin an open planting can be traced to cross-pollination, i.e. less thanone percent of soybean seed formed in an open planting is capable ofproducing F₁ hybrid soybean plants, See Jaycox, “EcologicalRelationships between Honey Bees and Soybeans,” appearing in theAmerican Bee Journal Vol. 110(8): 306-307 (August 1970). Thusintervention for control of pollination is critical to establishment ofsuperior varieties.

A cross between two different homozygous lines produces a uniformpopulation of hybrid plants that may be heterozygous for many gene loci.A cross of two plants each heterozygous at a number of gene loci willproduce a population of hybrid plants that differ genetically and willnot be uniform. Regardless of parentage, plants that have beenself-pollinated and selected for type for many generations becomehomozygous at almost all gene loci and produce a uniform population oftrue breeding progeny.

Soybeans, (Glucine max), can be bred by both self-pollination andcross-pollination techniques. Choice of breeding or selection methodsdepends on the mode of plant reproduction, the heritability of thetrait(s) being improved, and the type of variety used commercially(e.g., F₁ hybrid variety, pureline variety, etc.). For highly heritabletraits, a choice of superior individual plants evaluated at a singlelocation will be effective, whereas for traits with low heritability,selection should be based on mean values obtained from replicatedevaluations of families of related plants. Popular selection methodscommonly include pedigree selection, modified pedigree selection, massselection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Pedigree breeding and recurrent selection breeding methods are used todevelop varieties from breeding populations. Pedigree breeding startswith the crossing of two genotypes, each of which may have one or moredesirable characteristics that is lacking in the other or whichcomplements the other. If the two original parents do not provide allthe desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive generations. In the succeeding generationsthe heterozygous condition gives way to homogeneous lines as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding five or more generations of selfing and selection is practiced:F₁→F₂; F₂→F₃;F₃→F₄; F₄→F₅, etc.

Pedigree breeding is commonly used for the improvement ofself-pollinating crops. Two parents that possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁'s or by intercrossing two F₁'s(sib mating). Selection of the best individuals may begin in the F₂population; then, beginning in the F₃, the best individuals in the bestfamilies are selected. Replicated testing of families can begin in theF₄ generation to improve the effectiveness of selection for traits withlow heritability. At an advanced stage of inbreeding (i.e., F₆ and F₇),the best lines or mixtures of phenotypically similar lines are testedfor potential release as new varieties.

Backcross breeding has been used to transfer genes for simply inherited,highly heritable traits into a desirable homozygous variety or inbredline that is utilized as the recurrent parent. The source of the traitsto be transferred is called the donor parent. After the initial cross,individuals possessing the desired traits of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., variety) and the desirable traits transferred from thedonor parent. This approach has been used extensively for breedingdisease resistant varieties.

Each soybean breeding program should include a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goal and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful varieties produced per unit of input (e.g., per year, perdollar expended, etc.).

Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial varieties; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

Publically available or newly-released varieties of soybean can also beused as parental lines or starting materials for breeding or as sourcepopulations from which to develop or derive other soybean varieties orbreeding lines. These varieties or lines derived from publicallyavailable or newly-released varieties can be developed by using breedingmethods described earlier, such as pedigree breeding, backcrossing andrecurrent selection. As an example, when backcross breeding is used tocreate these derived lines or varieties in a soybean breeding program,publicly available or newly released varieties of soybeans can be usedas a parental line or starting material or source population and canserve as either the donor or recurrent parent. See for example, Fehr,“Breeding Methods for Cultivar Development”, Chapter 7, SoybeansImprovement, Production and Uses, 2^(nd) ed., Wilcox ed. 1987.

These processes, which lead to the final step of marketing anddistribution, can take from eight to twelve years from the time thefirst cross is made. Therefore, development of new varieties is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardvariety. Generally a single observation is inconclusive, so replicatedobservations are required to provide a better estimate of its geneticworth.

In addition to the preceding problem, it is not known how the genotypewill react with the environment. This genotype by environmentinteraction is an important, yet unpredictable, factor in plantbreeding. A breeder of ordinary skill in the art cannot predict thegenotype, how that genotype will interact with various environments orthe resulting phenotypes of the developing lines, except perhaps in avery broad and general fashion. A breeder of ordinary skill in the artwould also be unable to recreate the same line twice from the very sameoriginal parents, as the breeder is unable to direct how the genomescombine or how they will interact with the environmental conditions.This unpredictability results in the expenditure of large amounts ofresearch resources in the development of a superior new soybean variety.

The goal of soybean breeding is to develop new, unique and superiorsoybean varieties. In practical application of a chosen soybean breedingprogram, the breeder initially selects and crosses two or more parentallines, followed by repeated selfing and selection, producing many newgenetic combinations. The breeder can theoretically generate billions ofdifferent genetic combinations via crossing, selfing and mutations. Thebreeder has no direct control at the cellular level. Therefore, twobreeders will never develop the same line, or even very similar lines,having the same soybean traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made during and at the end of the growing season. The varietieswhich are developed are unpredictable for the reasons already mentioned.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinated crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect major faults and establish the level ofsuperiority or improvement over current varieties. In addition toshowing superior performance, there must be a demand for a new variety.The new variety must be compatible with industry standards, or mustcreate a new market. The introduction of a new variety may incuradditional costs to the seed producer, the grower, processor andconsumer, for special advertising and marketing, altered seed andcommercial production practices, and new product utilization. Thetesting preceding release of a new variety should take intoconsideration research and development costs as well as technicalsuperiority of the final variety. For seed-propagated varieties, it mustbe feasible to produce seed easily and economically.

Soybean (Glucine max), is an important and valuable field crop. Thus, acontinuing goal of soybean breeders is to develop stable, high yieldingsoybean varieties that are agronomically sound. The reasons for thisgoal are obviously to maximize the amount of grain produced on the landused and to supply food for both animals and humans. To accomplish thisgoal, the soybean breeder must select and develop soybean plants thathave the traits that result in superior varieties.

Pioneer soybean research staff create over 500,000 potential newvarieties each year. Of those new varieties, less than 50 and morecommonly less than 25 are actually selected for commercial use.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel soybean variety,designated 93B47. This invention thus relates to the seeds of soybeanvariety 93B47, to the plants of soybean 93B47 and to methods forproducing a soybean plant produced by crossing soybean variety 93B47with itself or another soybean plant, and the creation of variants bymutagenesis or transformation of soybean 93B47. This invention alsorelates to methods for producing other soybean varieties or breedinglines derived from soybean variety 93B47 and to soybean varieties orbreeding lines produced by those methods. Soybean variety 93B47 ischaracterized by a unique combination of yield potential, diseaseresistance and herbicide resistance for its maturity.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

B/A=BUSHELS PER ACRE. The seed yield in bushels/acre is the actual yieldof the grain at harvest.

BSR=BROWN STEM ROT TOLERANCE. This is a visual disease score from 1 to 9comparing all genotypes in a given test. The score is based on leafsymptoms of yellowing and necrosis caused by brown stem rot. A score of9 indicates no symptoms. Visual scores range down to a score of 1 whichindicates severe symptoms of leaf yellowing and necrosis.

CNKR=STEM CANKER TOLERANCE. This is a visual disease score from 1 to 9comparing all genotypes in a given test. The score is based uponpremature plant death. A score of 9 indicates no symptoms, whereas ascore of 1 indicates the entire experimental unit died very early.

COTYLEDON. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

EMBRYO. The embryo is the small plant contained within a mature seed.

F₃. This symbol denotes a generation resulting from the selfing of theF₂ generation along with selection for type and rogueing of off-types.The “F” number is a term commonly used in genetics, and designates thenumber of the filial generation. The “F₃” generation denotes theoffspring resulting from the selfing or self mating of members of thegeneration having the next lower “F” number, viz. the F₂ generation.

FECL=IRON-DEFICIENCY CHLOROSIS. Plants are scored 1 to 9 based on visualobservations. A score of 1 indicates the plants are dead or dying fromiron-deficiency chlorosis, a score of 5 means plants have intermediatehealth with some leaf yellowing and a score of 9 means no stunting ofthe plants or yellowing of the leaves.

FEY=Frogeye Tolerance. This is a visual disease score from 1 to 9comparing all genotypes in a given test. The score is based upon leaflesions. A score of 9 indicates no lesions, whereas a score of 1indicates severe leaf necrosis.

HABIT. This refers to the physical appearance of a plant. It can beeither determinate or indeterminate. In soybeans, indeterminatevarieties are those in which stem growth is not limited by formation ofa reproductive structure (i.e., flowers, pods and seeds) and hencegrowth continues throughout flowering and during part of pod filling.The main stem will develop and set pods over a prolonged period underfavorable conditions. In soybeans, determinate varieties are those inwhich stem growth ceases at flowering time. Most flowers developsimultaneously, and most pods fill at approximately the same time.

HGT=Plant Height. Plant height is taken from the top of the soil to toppod of the plant and is measured in inches.

HILUM. This refers to the scar left on the seed which marks the placewhere the seed was attached to the pod prior to it (the seed) beingharvested.

HYPL=HYPOCOTYL ELONGATION. This score indicates the ability of the seedto emerge when planted 3″ deep in sand and with a controlled temperatureof 25° C. The number of plants that emerge each day are counted. Basedon this data, each genotype is given a 1 to 9 score based on its rate ofemergence and percent of emergence. A score of 9 indicates an excellentrate and percent of emergence, an intermediate score of 5 indicatesaverage ratings and a 1 score indicates a very poor rate and percent ofemergence.

HYPOCOTYL. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

LDG=LODGING RESISTANCE. Lodging is rated on a scale of 1 to 9. A scoreof 9 indicates erect plants. A score of 5 indicates plants are leaningat a 45° angle in relation to the ground and a score of 1 indicatesplants are laying on the ground.

LEAFLETS. These are part of the plant shoot, and they manufacture foodfor the plant by the process of photosynthesis.

LLE=Linoleic Acid Percent. Linoleic acid is one of the five mostabundant fatty acids in soybean seeds. It is measured by gaschromatography and is reported as a percent of the total oil content.

LLN=Linolenic Acid Percent. Linolenic acid is one of the five mostabundant fatty acids in soybean seeds. It is measured by gaschromatography and is reported as a percent of the total oil content.

MAT ABS=ABSOLUTE MATURITY. This term is defined as the length of timefrom planting to complete physiological development (maturity). Theperiod from planting until maturity is reached is measured in days,usually in comparison to one or more standard varieties. Plants areconsidered mature when 95% of the pods have reached their mature color.

MATURITY GROUP. This refers to an agreed-on industry division of groupsof varieties, based on the zones in which they are adapted primarilyaccording to day length or latitude. They consist of very long daylength varieties (Groups 000, 00, 0), and extend to very short daylength varieties (Groups VII, VIII, IX, X).

OIL=Oil Percent. Soybean seeds contain a considerable amount of oil. Oilis measured by NIR spectrophotometry, and is reported on an as ispercentage basis.

OLC=OLEIC ACID PERCENT. Oleic acid is one of the five most abundantfatty acids in soybean seeds. It is measured by gas chromatography andis reported as a percent of the total oil content.

PLM=Palmitic Acid Percent. Palmitic acid is one of the five mostabundant fatty acids in soybean seeds. It is measured by gaschromatography and is reported as a percent of the total oil content.

POD. This refers to the fruit of a soybean plant. It consists of thehull or shell (pericarp) and the soybean seeds.

PRT=PHYTOPHTHORA TOLERANCE. Tolerance to Phytophthora root rot is ratedon a scale of 1 to 9, with a score of 9 being the best or highesttolerance ranging down to a score of 1 which indicates the plants haveno tolerance to Phytophthora.

PRM=Predicted Relative Maturity. Soybean maturities are divided intorelative maturity groups. In the United States the most common maturitygroups are 0 through VIII. Within these maturity groups the industrygenerally divides maturities into tenths of a relative maturity group.Within narrow comparisons, the difference of a tenth of a relativematurity group equates very roughly to a day difference in maturity atharvest.

PRO=Protein Percent. Soybean seeds contain a considerable amount ofprotein. Protein is generally measured by NIR spectrophotometry, and isreported on an as is percentage basis.

PUBESCENCE. This refers to a covering of very fine hairs closelyarranged on the leaves, stems and pods of the soybean plant.

RKA=Root-knot nematode, peanut. This is a visual disease score from 1 to9 comparing all genotypes in a given test. The score is based upondigging plants to look at the roots for presence or absence of galling.A score of 9 indicates that there is no galling of the roots, a score of1 indicates large severe galling cover most of the root system whichresults in pre-mature death from decomposing of the root system.

RKI=Root-knot nematode, southern. This is a visual disease score from 1to 9 comparing all genotypes in a given test. The score is based upondigging plants to visually score the roots for presence or absence ofgalling. A score of 9 indicates that there is no galling of the roots, ascore of 1 indicates large severe galling cover most of the root systemwhich results in pre-mature death from decomposing of the root system.

SDS=Sudden Death Syndrome. Tolerance to Sudden Death Syndrome is ratedon a scale of 1 to 9, with a score of 1 being very susceptible rangingup to a score of 9 being resistant.

SILB=Seeds per Pound. Soybean seeds vary in seed size, therefore, thenumber of seeds required to make up one pound also varies. This affectsthe pounds of seed required to plant a given area, and can also impactend uses.

SH=SHATTERING. This refers to the amount of pod dehiscence prior toharvest. Pod dehiscence involves seeds falling from the pods to thesoil. This is a visual score from 1 to 9 comparing all genotypes withina given test. A score of 9 means pods have not opened and no seeds havefallen out. A score of 5 indicates approximately 50% of the pods haveopened, with seeds falling to the ground and a score of 1 indicates 100%of the pods are opened.

SHOOTS. These are a portion of the body of the plant. They consist ofstems, petioles and leaves.

STC=STEARIC ACID PERCENT. Stearic acid is one of the five most abundantfatty acids in soybean seeds. It is measured by gas chromatography andis reported as a percent of the total oil content.

WH MD=WHITE MOLD TOLERANCE. This is a visual disease score from 1 to 9comparing all genotypes in a given test. The score is based uponobservations of mycelial growth and death of plants. A score of 9indicates no symptoms. Visual scores of 1 indicate complete death of theexperimental unit.

DETAILED DESCRIPTION OF THE INVENTION

A soybean variety needs to be highly homogeneous, homozygous andreproducible to be useful as a commercial variety. There are manyanalytical methods available to determine the homozygotic and phenotypicstability of these inbred lines.

The oldest and most traditional method of analysis is the observation ofphenotypic traits. The data is usually collected in field experimentsover the life of the soybean plants to be examined. Phenotypiccharacteristics most often observed are for traits associated with seedyield, seed protein and oil content, lodging resistance, diseaseresistance, maturity, plant height, and shattering.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), and Simple SequenceRepeats (SSRs) which are also referred to as Microsatellites.

The variety of the invention has shown uniformity and stability for alltraits, as described in the following variety description information.It has been self-pollinated a sufficient number of generations, withcareful attention to uniformity of plant type to ensure homozygosity andphenotypic stability. The line has been increased with continuedobservation for uniformity. No variant traits have been observed or areexpected in 93B47, as described in Table 1 (Variety DescriptionInformation).

Soybean variety 93B47 is a white flowered, soybean variety with tawnypubescence, brown hila, and a relative maturity of 34. 93B47 alsodemonstrates very good yield in combination with Multi-race Phytophthoraresistance (Rps1c). Variety 93B47 further demonstrates very goodtolerance to Brown Stem Rot and displays a substantial degree ofglyphosate resistance. 93B47 exhibits good iron deficiency chlorosis andis particularly suited to the Plains, Southern Plains and Easternregions of the United States including Iowa, Illinois, Indiana,Missouri, Michigan and Ohio. This variety does well in Phytophthoramegasperma infected soils where the Rps1c gene provides resistance.

Soybean variety 93B47, being substantially homozygous, can be reproducedby planting seeds of the line, growing the resulting soybean plantsunder self-pollinating or sib-pollinating conditions, and harvesting theresulting seed, using techniques familiar to the agricultural arts.

TABLE 1 VARIETY DESCRIPTION INFORMATION 93B47 A. Mature SeedCharacteristics: Seed Coat Color: yellow Seed Coat Luster: shiny SeedSize (grams per 100 seeds): 14 Hilum Color: brown B. Leaf: LeafletShape: ovate Leaf Color: dark green C. Plant Characteristics: FlowerColor: white Pod Color: tan Plant Pubescence Color: tawny Plant Habit:indeterminate Maturity Group: III Maturity Sub-Group: 4 D. FungalDiseases (S = susceptible R = resistant) Brown Stem Rot (Cephalosporiumgregatum): very good resistance Phytophthora Rot (Phytophthoramegasperma var. sojae): Race 1: R Race 4: S Race 5: S Race 7: R E.Nematode Diseases (S = susceptible R = resistant) Soybean Cyst NematodeRace 3: S Soybean Cyst Other Specify: Race 14: moderately susceptibleIron Chlorosis: above average F. Submitted Seed Content (% Protein): 36Submitted Seed Content (% Oil): 20 Publications useful as references ininterpreting Table 1 include: Caldwell, B. E. ed. 1973. “Soybeans:Improvement, Production, and Uses” Amer. Soc. Agron. Monograph No. 16;Buttery, B. R., and R. I. Buzzell 1968. “Peroxidase Activity in Seed ofSoybean Varieties” Crop Sci. 8: 722-725; Hymowitz, T. 1973.“Electrophoretic analysis of SBTI-A2 in the USDA Soybean GermplasmCollection” Crop Sci., 13: 420-421; Payne R. C., and L.F. Morris, 1976.“Differentiation of Soybean Varieties by Seedling Pigmentation Patterns”J. Seed. Technol. 1: 1-19. The disclosures of which are eachincorporated by reference in their entirety.

FURTHER EMBODIMENTS OF THE INVENTION

Transformation of Soybean

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreign,additional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed soybean plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the soybean plant(s).

Expression Vectors for Soybean Transformation

Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e. inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80: 4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5: 299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210: 86 (1987), Svab etal., Plant Mol. Biol. 14: 197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or broxynil. Comai et al.,Nature 317: 741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618(1990) and Stalker et al., Science 242: 419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67(1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant CellRep. 8: 643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5: 387 (1987)., Teeri et al., EMBO J. 8: 343(1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3: 1681 (1984).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes Publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115: 15Ia (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263: 802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters. As used herein “promoter” includes referenceto a region of DNA upstream from the start of transcription and involvedin recognition and binding of RNA polymerase and other proteins toinitiate transcription. A “plant promoter” is a promoter capable ofinitiating transcription in plant cells. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, seeds, fibers,xylem vessels, tracheids, or sclerenchyma. Such promoters are referredto as “tissue-preferred”. Promoters which initiate transcription only incertain tissue are referred to as “tissue-specific”. A “cell type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression insoybean. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in soybean. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal. Plant Mol. Biol. 22: 361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al. PNAS 90: 4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243: 32-38 (1994)) or Tet repressor from Tn10 (Gatzet al., Mol. Gen. Genet. 227: 229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88: 0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression insoybean or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in soybean.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313: 810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12: 619-632 (1989) andChristensen et al., Plant Mol. Biol. 18: 675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81: 581-588 (1991)); MAS (Velten et al., EMBOJ. 3: 2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genet. 231: 276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, a XbaI/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence that has substantial sequencesimilarity to said XbaI/NcoI fragment), represents a particularly usefulconstitutive promoter. See PCT application WO96/30530.

C. Tissue-specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin soybean. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in soybean. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA82: 3320-3324 (1985)); a leaf-specific and light-induced promoter suchas that from cab or rubisco (Simpson et al., EMBO J. 4(11): 2723-2729(1985) and Timko et al., Nature 318: 579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genet. 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genet. 224: 161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6: 217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker et al., Plant Mol. Biol. 20: 49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes FromBarley”, Plant Mol. Biol. 9: 3-17 (1987), Lemer et al., PlantPhysiol.91: 124-129 (1989), Fontes et al., Plant Cell 3: 483-496 (1991),Matsuoka et al., Proc. Nat. Acad. Sci. 88: 834 (1991), Gould et al., J.Cell Biol 108: 1657 (1989), Creissen et al., Plant J. 2: 129 (1991),Kalderon, D., Robers, B., Richardson, W., and Smith A., “A short aminoacid sequence able to specify nuclear location”, Cell 39: 499-509(1984), Stiefel, V., Ruiz-Avila, L., Raz R., Valles M., Gomez J., PagesM., Martinez-Izquierdo J., Ludevid M., Landale J., Nelson T., andPuigdomenech P., “Expression of a maize cell wall hydroxyproline-richglycoprotein gene in early leaf and root vascular differentiation”,Plant Cell 2: 785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a soybean plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Glick and Thompson, METHODS IN PLANTMOLECULAR BIOLOGY AND BIOTECHNOLOGY 269-284 (CRC Press, Boca Raton,1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below.

1. Genes that Confer Resistance to Pests or Disease and That Encode

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

(B) A gene conferring resistance to a pest, such as soybean cystnematode. See e.g. PCT Application WO96/30517; PCT ApplicationWO93/19181.

(C) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 1.09 (1986), who disclose the cloning and nucleotide sequenceof a Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection (Manassas,Va.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

(D) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

(E) A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

(F) An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262: 16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21: 985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

(G) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(H) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163: 1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

(I) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116: 165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

(J) An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(K) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

(L) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(M) A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

(N) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(O) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(P) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(Q) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(R) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10: 1436 (1992). The cloning and characterization of a gene whichencodes a bean endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2: 367 (1992).

(S) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance to a Herbicide, for Example

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl.Genet. 80: 449(1990), respectively.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase, bar, genes), andpyridinoxy or phenoxy propionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes That Confer or Contribute to a Value-added Trait, Such as

(A) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Nat'l. Acad. Sci.USA 89: 2624 (1992).

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35: 383 (1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α-amylase), Elliot et al., Plant Molec.Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes),Søgaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102: 1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Soybean Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant. Sci. 10: 1 (1991). Descriptions of Agrobacterium vector systemsand methods for Agrobacterium-mediated gene transfer are provided byGruber et al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8: 238 (1989). See also, U.S. Pat. No. 5,563,055, (Townsend andThomas), issued Oct. 8, 1996.

B. Direct Gene Transfer

Several methods of plant transformation, collectively referred to asdirect gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes. Sanfordet al., Part. Sci. Technol. 5: 27 (1987), Sanford, J. C., TrendsBiotech. 6: 299 (1988), Klein et al., Bio/Technology 6: 559-563 (1988),Sanford, J. C., Physiol Plant 79: 206 (1990), Klein et al.,Biotechnology 10: 268 (1992). See also U.S. Pat. No. 5,015,580(Christou, et al), issued May 14, 1991; U.S. Pat. No. 5,322,783 (Tomes,et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.99: 161 (1985) and Draper et al., Plant Cell Physiol. 23: 451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24: 51-61 (1994).

Following transformation of soybean target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular soybean line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Tissue Culture of Soybeans

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybean,” Crop Sci.31:333-337 (1991); Stephens, P. A. et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. (1991)82:633-635; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glucine max (L.) Merr.,” Plant Cell, Tissueand Organ Culture, 28:103-113 (1992); Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glucine max L. Merr.):Genotypic Differences in Culture Response,” Plant Cell Reports (1992)11:285-289; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5 (1992); and Shetty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glucine max (Merrill.)by Allantoin and Amides,” Plant Science 81:(1992) 245-251; as well asU.S. Pat. No. 5,024,944, issued Jun. 18, 1991 to Collins et al. and U.S.Pat. No. 5,008,200, issued Apr. 16, 1991 to Ranch et al., thedisclosures of which are hereby incorporated herein in their entirety byreference. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce soybean plants having thephysiological and morphological characteristics of soybean variety93B47.

This invention also is directed to methods for producing a soybean plantby crossing a first parent soybean plant with a second parent soybeanplant wherein the first or second parent soybean plant is a soybeanplant of the variety 93B47. Further, both first and second parentsoybean plants can come from the soybean variety 93B47. Thus, any suchmethods using the soybean variety 93B47 are part of this invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using soybean variety 93B47 as a parent arewithin the scope of this invention, including those developed fromvarieties derived from soybean variety 93B47. Advantageously, thesoybean variety could be used in crosses with other, different, soybeanplants to produce first generation (F₁) soybean hybrid seeds and plantswith superior characteristics. The variety of the invention can also beused for transformation where exogenous genes are introduced andexpressed by the variety of the invention. Genetic variants createdeither through traditional breeding methods using variety 93B47 orthrough transformation of 93B47 by any of a number of protocols known tothose of skill in the art are intended to be within the scope of thisinvention.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which soybean plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, seed, flowers, pods,leaves, roots, root tips, anthers, and the like.

INDUSTRIAL APPLICABILITY

The seed of soybean variety 93B47, the plant produced from the seed, thehybrid soybean plant produced from the crossing of the variety with anyother soybean plant, hybrid seed, and various parts of the hybridsoybean plant can be utilized for human food, livestock feed, and as araw material in industry.

The soybean is the world's leading source of vegetable oil and proteinmeal. The oil extracted from soybeans is used for cooking oil,margarine, and salad dressings. Soybean oil is composed of saturated,monounsaturated and polyunsaturated fatty acids. It has a typicalcomposition of 11% palmitic, 4% stearic, 25% oleic, 50% linoleic and 9%linolenic fatty acid content (“Economic Implications of Modified SoybeanTraits Summary Report”, Iowa Soybean Promotion Board & American SoybeanAssociation Special Report 92S, May 1990). Changes in fatty acidcomposition for improved oxidative stability and nutrition areconstantly sought after. Industrial uses of soybean oil which issubjected to further processing include ingredients for paints,plastics, fibers, detergents, cosmetics, and lubricants. Soybean oil maybe split, inter-esterified, sulfurized, epoxidized, polymerized,ethoxylated, or cleaved. Designing and producing soybean oil derivativeswith improved functionality, oliochemistry, is a rapidly growing field.The typical mixture of triglycerides is usually split and separated intopure fatty acids, which are then combined with petroleum-derivedalcohols or acids, nitrogen, sulfonates, chlorine, or with fattyalcohols derived from fats and oils.

Soybean is also used as a food source for both animals and humans.Soybean is widely used as a source of protein for animal feeds forpoultry, swine and cattle. During processing of whole soybeans, thefibrous hull is removed and the oil is extracted. The remaining soybeanmeal is a combination of carbohydrates and approximately 50% protein.

For human consumption soybean meal is made into soybean flour which isprocessed to protein concentrates used for meat extenders or specialtypet foods. Production of edible protein ingredients from soybean offersa healthy, less expensive replacement for animal protein in meats aswell as dairy-type products.

PERFORMANCE EXAMPLES OF 93B47

In the examples that follow, the traits and characteristics of soybeanvariety 93B47 are given in paired comparison with another soybeanvariety grown in the environment under the same conditions. The datacollected on each soybean variety is presented for key characteristicsand traits.

The results in Table 2A compare variety 93B47 to another similarlyadapted Pioneer soybean variety, 92B91. The results indicate thatvariety 93B47 is significantly higher yielding than variety 92B91.Variety 93B47 is also later to mature with a significantly higherabsolute maturity and predicted relative maturity score than variety92B91. Variety 93B47 further exhibits significantly superior resistanceto plant lodging than variety 92B91.

The results in Table 2B compare variety 93B47 to another similarlyadapted Pioneer soybean variety, 93B26. According to the results,variety 93B47 is significantly higher yielding than variety 93B26.Variety 93B47 is also later to mature with a significantly higherabsolute maturity and predicted relative maturity score than variety93B26. Variety 93B47 further exhibits significantly superior resistanceto plant lodging and a larger seed size with a significantly lowernumber of seeds per pound than variety 93B26. Variety 93B47 shows asomewhat higher susceptibility to Sudden Death syndrome than variety93B26.

The results in Table 2C compare variety 93B47 to another similarlyadapted Pioneer soybean variety, 93B35. The results indicate thatvariety 93B47 is significantly higher yielding than variety 93B35.Variety 93B47 exhibits a significantly higher predicted relativematurity score and a significantly taller plant stature than variety93B35. Variety 93B47 further exhibits a somewhat higher susceptibilityto plant lodging than variety 93B35.

The results in Table 2D compare variety 93B47 to another similarlyadapted Pioneer soybean variety, 93B51. According to the table variety93B47 is significantly higher yielding than variety 93B51. Variety 93B47is also earlier to mature with a significantly lower absolute maturityand predicted relative maturity score than variety 93B51. Variety 93B47further exhibits significantly higher seed protein content than variety93B51.

The results in Table 2E compare variety 93B47 to another similarlyadapted Pioneer soybean variety, 93B53. The table shows that variety93B47 is significantly higher yielding than variety 93B53. Variety 93B47exhibits a significantly taller plant stature with a somewhat highersusceptibility to lodging than variety 93B53. Variety 93B47 furtherexhibits significantly superior tolerance to Sudden Death Syndrome thanvariety 93B53.

The results in Table 2F compare variety 93B47 to another similarlyadapted Pioneer soybean variety, 93B84. According to the results,variety 93B47 is significantly higher yielding than variety 93B84.Variety 93B47 is also earlier to mature with a significantly lowerabsolute maturity and predicted relative maturity score than variety93B84. Variety 93B47 further exhibits significantly shorter plantstature with a somewhat higher susceptibility to plant lodging thanvariety 93B84. Variety 93B47 exhibits a somewhat higher susceptibilityto Brown Stem Rot than variety 93B84

The results in Table 2G compare variety 93B47 to another similarlyadapted Pioneer soybean variety, AG3302. According to the results,variety 93B47 exhibits a significantly lower absolute maturity scorethan variety AG3302. The table has few repetitions of data for fewtraits, it is expected that other significant differences will beobserved with additional data.

The results in Table 2H compare variety 93B47 to another similarlyadapted Asgrow soybean variety, AG3303. The table shows that variety93B47 exhibits a significantly lower absolute maturity score thanvariety AG3303. Variety 93B47 also shows a somewhat highersusceptibility to lodging than variety AG3303.

The results in Table 2I compare variety 93B47 to another similarlyadapted Pioneer soybean variety, 93B01. The table indicates that variety93B47 is later to mature with a significantly higher absolute maturityand predicted relative maturity score than variety 93B01. Variety 93B47further exhibits significantly taller plant stature with a somewhathigher susceptibility to plant lodging than variety 93B01. Variety 93B47shows a significantly lower seed oil content than variety 93B01.

TABLE 2A PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = 92B91MAT PRM VAR B/A ABS MAT HGT LDG PRO OIL 93- 56.7 125.5 33 38 7 40.8 22.8B47 92- 51.5 123 30 39.1 4.5 39.9 23.5 B91 REPS 15 8 4 8 4 4 4 DIFF 5.32.5 2 1.1 2.5 0.9 0.7 PRO- .032+ .007# .014+ 0.363 .015+ 0.308 0.216 BS/L FEC VAR B BSR L 93B47 3075.6 7 6.7 92B91 3160.6 9 6.7 REPS 4 1 1DIFF 84.9 2 0 PROB 0.305 * = significant at the 10% level +significantat the 5% level #significant at the 1% level

TABLE 2B PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = 93B26MAT PRM VAR B/A ABS MAT HGT LDG PRO OIL 93- 49.6 130.9 34 36 6.8 40.521.7 B47 93- 46.6 129.4 33 34.8 6 40.6 20.8 B26 REPS 44 20 2 14 12 3 3DIFF 3 1.6 1 1.2 0.7 0.1 0.9 PRO- .011+ .005# .036+ 0.202 .040+ 0.9090.175 B S/L FEC VAR B BSR L SDS 93B47 2966 6 6.5 4.8 93B26 3311.9 7.5 67.8 REPS 3 2 1 6 DIFF 345.9 1.5 0.5 3.1 PROB .000# 0.205 .039+ * =significant at the 10% level +significant at the 5% level #significantat the 1% level

TABLE 2C PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = 93B35MAT PRM VAR B/A ABS MAT HGT LDG PRO OIL 93- 49.9 131.4 34 33.8 6.8 40.521.7 B47 93- 46.2 130.9 33 28.8 8.3 41.5 21.9 B35 REPS 83 36 9 28 12 3 3DIFF 3.7 0.5 1 4.9 1.5 1 0.3 PRO- .000# .061* .024+ .000# .002# 0.2420.371 B S/L FEC VAR B BSR L SDS 93B47 2966 6.5 6.5 5.3 93B35 3137.3 6.63 6 REPS 3 8 1 12 DIFF 171.3 0.1 3.5 0.7 PROB 0.373 0.844 0.231*significant at the 10% level +significant at the 5% level #significantat the 1% level

TABLE 2D PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = 93B51MAT PRM VAR B/A ABS MAT HGT LDG PRO OIL 93- 51.4 129.4 33 36.7 6.8 40.722.3 B47 93- 49.7 131.1 36 36.4 6.6 39.4 22.1 B51 REPS 59 28 6 22 16 7 7DIFF 1.7 1.8 2 0.4 0.2 1.3 0.2 PRO- .021+ .000# .008# 0.593 0.625 .003#0.374 B S/L FEC VAR B BSR L SDS 93B47 3028.6 6.3 6.5 4.8 93B51 3145.87.7 5.5 4.6 REPS 7 3 1 6 DIFF 117.2 1.3 1 0.2 PROB 0.112 .057* 0.675 * =significant at the 10% level +significant at the 5% level #significantat the 1% level

TABLE 2E PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = 93B53MAT PRM VAR B/A ABS MAT HGT LDG PRO OIL 93- 49.2 130.3 34 34.1 6.9 40.521.7 B47 93- 47.5 131 35 32.1 7.9 40.3 21.8 B53 REPS 102 44 13 38 14 3 3DIFF 1.8 0.7 1 2 1 0.1 0.2 PRO- .007# 0.106 .056* .000# .005# 0.8160.596 B S/L FEC VAR B BSR L SDS 93B47 2966 6.3 6.5 6.5 93B53 3009.6 7.36.8 5 REPS 3 9 1 21 DIFF 43.6 1 0.3 1.4 PROB 0.632 .067* .000#*significant at the 10% level +significant at the 5% level #significantat the 1% level

TABLE 2F PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = 93B84MAT PRM VAR B/A ABS MAT HGT LDG PRO OIL 93- 47.7 129.9 35 34.7 6.9 40.521.7 B47 93- 45.7 133.5 38 35.7 8.1 41.9 21.3 B84 REPS 85 35 9 32 16 3 3DIFF 2 3.6 3 1 1.2 1.4 0.4 PRO- .011+ .000# .000# .023+ .003# .085*0.147 B S/L VAR B BSR SDS 93B47 2966 6.8 6.5 93B84 3066.7 9 6 REPS 3 524 DIFF 100.6 2.2 0.5 PROB 0.394 .020+ 0.347 *significant at the 10%level +significant at the 5% level #significant at the 1% level

TABLE 2G PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = AG3302MAT PRM VAR B/A ABS MAT HGT LDG BSR SDS 93B47 48.4 131.7 33 35.5 6.3 6 6AG3302 51.8 133.6 34 37.9 5.6 9 6.3 REPS 22 9 1 6 5 1 3 DIFF 3.3 1.8 12.4 0.7 3 0.3 PROB .078* .008# .085* 0.597 0.808 *significant at the 10%level +significant at the 5% level #significant at the 1% level

TABLE 2H PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = AG3303MAT PRM VAR B/A ABS MAT HGT LDG SDS 93B47 48.7 131.7 33 35.5 6.3 6AG3303 47.2 135.1 36 38.1 8.2 6.3 REPS 22 9 1 6 5 3 DIFF 1.5 3.4 2 2.61.9 0.3 PROB 0.16 .001# 0.166 .009# 0.84 * = significant at the 10%level +significant at the 5% level #significant at the 1% level

TABLE 2I PAIRED COMPARISON REPORT VARIETY #1 = 93B47 VARIETY #2 = 93B01MAT PRM VAR B/A ABS MAT HGT LDG PRO OIL 93- 50 131.3 34 33.5 6.8 40.521.7 B47 93- 48.6 127.5 30 28.1 8.4 40.2 22.7 B01 REPS 88 38 10 30 12 33 DIFF 1.3 3.8 4 5.4 1.6 0.2 1 PRO- .061* .000# .000# .000# .001# 0.551.042+ B S/L FEC VAR B BSR L SDS 93B47 2966 63 6.5 5.3 93B01 3380.4 6 65.8 REPS 3 9 1 12 DIFF 414.4 0.3 0.5 0.4 PROB .062* 0.5 0.426*significant at the 10% level +significant at the 5% level #significantat the 1% level

DEPOSITS

Applicant(s) has made a deposit of at least 2500 seeds of Soybeanvariety 93B47 with the American Type Culture Collection (ATCC),Manassas, Va. 20110 USA, ATCC Deposit No. PTA-4502. The seeds depositedwith the ATCC on Jun. 26, 2002 were taken from the deposit maintained byPioneer Hi-Bred International, Inc., 800 Capital Square, 400 LocustStreet, Des Moines, Iowa 50309-2340, since prior to the filing date ofthis application. Access to this deposit will be available during thependency of the application to the Commissioner of Patents andTrademarks and persons determined by the Commissioner to be entitledthereto upon request. Upon allowance of any claims in the application,the Applicant(s) will make available to the public pursuant to 37 CFR1.808(2), a deposit of at least 2500 seeds of variety 93B47 with theAmerican Tissue Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va.; 20110-2209. This deposit of Soybean variety 93B47 will bemaintained in the ATCC Depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theenforceable life of the Pat., whichever is longer, and will be replacedif it ever becomes nonviable during that period. Additionally,Applicant(s) have or will satisfy all the requirements of 37 C.F.R. §§1.801-1.809, including providing an indication of the viability of thesample. Applicant(s) has no authority to waive any restrictions imposedby law on the transfer of biological material or its transportation incommerce. Applicant(s) does not waive any infringement of its rightsgranted under this patent or under the Plant Variety Protection Act (7USC 2321 et seq.).

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be obvious that certain changes and modifications suchas single gene modifications and mutations, somoclonal variants, variantindividuals selected from large populations of the plants of the instantvariety and the like may be practiced within the scope of the invention,as limited only by the scope of the appended claims.

What is claimed is:
 1. A soybean seed designated 93B47, representativeseed of said soybean variety 93B47 having been deposited under ATCCAccession No. PTA-4502.
 2. A soybean plant, or a part thereof, producedby growing the seed of claim
 1. 3. The soybean plant part of claim 2wherein said part is pollen.
 4. The soybean plant part of claim 2wherein said part is an ovule.
 5. A tissue culture of protoplasts orregenerable cells from the plant of claim
 2. 6. The tissue cultureaccording to claim 5, the cells or protoplasts of the tissue culture areobtained from plant tissues selected from the group consisting of: leaf,pollen, cotyledon, hypocotyl, embryos, root, pod, flower, shoot andstalk.
 7. A soybean plant regenerated from the tissue culture of claim5, having all the morphological and physiological characteristics ofsoybean variety 93B47, representative seed of said soybean variety 93B47having been deposited under ATCC Accession No. PTA-4502.
 8. A method forproducing a progeny soybean plant comprising crossing the plant of claim2 with a second soybean plant, harvesting the resultant soybean seed,and growing a progeny soybean plant.